This continuation project proposes extensive further studies on elastin biology and biochemistry. These studies are designed to delineate the molecular events of elastogenesis in physiologic situations concentrating on regulation of elastin gene expression both at transcriptional and post- transcriptional levels. This proposal is based on our significant progress made on molecular biology of elastin during the previous project period. The progress made includes (a) development and sequencing of full-length human elastin cDNAs; (b) characterization of the entire elastin gene structure with respect to exon/intron organization; (c) elucidation of alternative splicing occuring during post-transcriptional processing of elastin primary RNA transcripts; (d) characterization of structural features, including the precise nucleotide sequence, of ~ 2.2 kb of 5'- flanking region of the elastin gene containing the putative promoter elements; (e) development of functional elastin promoter/CAT gene constructs, and demonstration of functional promoter activity in a variety of human and rodent cell lines, including human skin fibroblasts; (f) construction of 5'-3' and 3'-5' deletion libraries that have delineated the basic promoter element and identified several positive (enhancer-like) and negative (down-regulatory/silencer) upstream elements. The specific technologies that the proposed research will utilize include (a) Northern and slot-blot hybridizations with sequence-specific cDNAs and synthetic oligomers; (b) polymerase chain reaction amplification for identification of human elastin cDNA and mRNA sequences; (c) transient cell transient cell transfection assays utilizing promoter/CAT gene constructs for elucidation of cis-and trans-acting control elements in human cells. We expect that the approaches utilizing these highly sophisticated techniques of molecular biology will allow us to pinpoint the key regulatory elements controlling elastogenesis in tissues under physiologic conditions. Elucidation of normal biochemistry and molecular biology of elastin provides a basis to explore elastin aberrations in clinical situations in the future.